1. Field of the Invention
The present invention relates to methods, reagents and kits for the determination of the presence of target nucleotide sequences. In particular, the present invention relates to a method for conducting an assay for the presence of a specific target nucleotide sequence in a sample containing an unknown quantity of said specific target nucleotide sequence and to probes for use in such an assay.
2. Description of the Prior Art
In the technology of manipulating genetic material or in evaluating the genetic character of an organism, it is often desirable to ascertain if a particular gene or part of a gene is present in an organism or in an extracellular extract of genetic material from that organism. Since any gene or gene portion is, in essence, a specific sequence of nucleotide bases forming all or part of a polynucleotide molecule, it is possible to directly test the sample polynucleotide to discover if the specific sequence of nucleotide bases forming the gene is present in the sample.
Interest in specific sequences of nucleotide bases may involve detecting the presence of pathogens, determining the presence of alleles, detecting the presence of lesions in a host genome and detecting a particular mRNA or the modification of a cellular host, to name only a few illustrative examples. Genetic diseases such as Huntington chorea, muscular dystrophy, phenylketonuria, thalassemias and sickle cell anemia can be diagnosed by the analysis of an individual's DNA. Furthermore, diagnosis or identification of viruses, viroids, bacteria, fungi, protozoa or any other plant or animal life form can be determined by hybridization assays with nucleotide probes.
Nucleic acid detection assays of various types have been documented in the literature. These types of assays, and in particular those requiring detection of polynucleotides, are based on the purine-pyrimidine base pairing properties of complementary nucleic acid strands in DNA-DNA or DNA-RNA duplexes This base-pairing process most frequently occurs through formation of hydrogen bonds in the pairing of adenosine-thymine (A-T) and guanosine-cytosine (G-C) bases in double-stranded DNA; adenosine-uracil base pairs may additionally be formed by hydrogen bonding in DNA-RNA hybrid molecules. Base pairing of nucleic acid strands for determination of the presence or absence of a given nucleotide sequence involving sample nucleotide sequences and a probe nucleotide sequence is commonly referred to as nucleic acid hybridization or simply hybridization.
One of the most powerful tools of molecular biology is the ability to fractionate nucleic acids and to determine which nucleic acids have sequences complementary to an array of DNA or RNA molecules. The Southern blot is a well known method for transferring electrophoretically fractioned DNA from a gel matrix to a nitrocellulose solid support by passive diffusion, followed by hybridization to a labeled probe. Similar procedures are used for detecting RNA with minor modifications and this method is known in the art as the Northern blot. The use of dried agarose gels as the immobilized phase is known as the Unblot method. All of these assay techniques are valuable tools for analyzing mRNA's, clones, genes, fragments, flanking sequences, repetitive elements and the like.
U.S. Pat. No. 4,358,535 describes a method for detecting pathogens using a target nucleic acid sequence. The method involves fixing a target nucleic acid sequence to an inert support before hybridization with a radioactively labeled nucleotide probe. The target nucleic acid sequence is then determined by detecting the presence of any label on the inert support.
European Patent Application No. 0 117 440 discloses non-radioactive chemically labeled polynucleotide probes and methods of using the probes. The target nucleic acid sequence is also fixed to a solid support.
U.S. Pat. Nos. 4,767,699 and 4,795,701 disclose a nucleic acid displacement assays. These assays utilize two polynucleotides; one polynucleotide is labeled, and the other polynucleotide is used to displace the labeled probe from the target sequence, thereby allowing detection of the target molecule. These assays use ATP to detect whether hybridization has occurred with the target molecule.
Many of the assays using nucleotide probes have problems in the detection systems. Sensitivity of the labeled probe and background levels that are generated during the assay often lead to erroneous results.
To facilitate more efficient detection of a nucleic acid sequence from a given sequence of DNA or RNA a target amplification method may be utilized. This method, known as PCR, is described in U.S. Pat. No, 4,683,195 and uses a set of primers and a DNA polymerase to extend the nucleic acid sequence of the target nucleotide and amplify it for future probe detection. By amplifying the DNA sequence, the target nucleotide can be more efficiently detected with the nucleotide probe. One of the problems encountered in this probe assay is contamination of the reaction medium.
Type II restriction enzymes are known in the art for making double-stranded scissions at specific sites within a DNA molecule. These enzymes are prevalent in bacteria, contain only one type of subunit and Mg.sup.2+ alone is required for DNA cleavage. DNA cleavage or scission occurs at specific sites within or adjacent to the enzyme's recognition site. More than 400 restriction enzymes have been isolated from bacterial strains to date. These restriction enzymes have been characterized primarily with respect to their recognition sequences and cleavage specificity. The majority of restriction enzymes or endonucleases recognize sequences 4-6 nucleotides in length, but some have been found with 7-8 base recognition sites. Most, but not all, recognition sites contain a dyad axis of symmetry and in most cases all the bases within the site are uniquely specified. Recognition of the symmetrical sequence of the hybridized sequences or palindromes is made by endonucleases. Endonucleases with symmetrical recognition sites generally cleave symmetrically.
The use of restriction enzymes with their specific cleavage sites is well recognized in the art. Usually restriction enzymes are used for the specific mapping, cloning and characterization of DNA sequences. However, they have been used in various nucleotide probe assays. For instance, U.S. Pat. No. 4,683,194 discloses a method for detecting the presence or absence of a specific restriction site in a nucleic acid sequence by hybridization with a nucleic acid probe that is complementary to one strand of the nucleic acid sequence spanning the restriction site. The hybridized sequence is then cleaved with a restriction enzyme and the resulting cut and uncut oligomers are separated and detected based on the type of probe label.
A similar concept for detecting a target nucleotide having a half-restriction site is set forth in U.S. Pat. No. 4,725,537. This patent discloses the use of a restriction endonuclease in a displacement-type of assay.
Another type of assay that uses the concept of cleaving a nucleic acid sequence in a nucleotide probe is disclosed in U.S. Pat. No. 4,876,187. This method is used to detect DNA or RNA sequences by specifically cleaving the nucleic acid sequence of the probe at in least one point thereby removing any reporter molecules not bound to a complementary target DNA sequence. This assay improves the signal to noise ratio of the detection system and is a highly sensitive assay.
Although the aforementioned assays do provide a method for detecting nucleic acid sequences in a target molecule, the need still exists for an assay system that provides very high sensitivity, ease of detection, less contamination in the assay medium and ease of operation, while avoiding false positive results.
The present invention overcomes the disadvantages associated with the techniques discussed above by introducing a highly sensitive detection method for detecting a nucleic acid sequence through the use of a novel form of restriction amplification. A second oligonucleotide is present in the assay to recycle the cleaved target sequence of interest thereby amplifying the labelled and cleaved probe oligonucleotide.